1. bantam Encodes a Developmentally Regulated microRNA that Controls Cell Proliferation and Regulates the Proapoptotic Gene hid in Drosophila
Julius Brennecke, David Hipfner, Alexander Stark, Robert
Russell, and Stephen M. Cohen
Cell, Vol. 113, 25-36, April 4, 2003.
European Molecular Biology Laboratory, Heidelberg, Germany

2. Background Hipfner, Weigmann and Cohen, Genetics, 2002
The bantam Gene Regulates Drosophila Growth
performed a gain-of-function screen using the EP method
(Rorth, 1996)
screened for GAL4-dependent effects on tissue size
without disrupting pattern
identified a locus that they called bantam

3. Background continued
identified 3 EP lines in a 12 kb interval at 61C7-8
P-element-mediated excision of one EP was used to create
a deletion (21 kb)
deletion is homozygous lethal at early puapl stages;
mutant larvae lack detectable imaginal discs
Deletion/P allele animals are viable but 15% smaller
small size is due to a reduction in cell number, not cell size
overexpression of bantam causes overgrowth of tissue
( wing and eye)
no predicted genes within the surrounding 41 kb interval
Expression levels (by Northern) of neighbouring genes
appeared normal in the bantam deletion

4. Figure 1. Map of the bantam Locus
41 kb region
(btn predicted genes)
bantam
hypomorph
Overlap region
BLAST:
Drosophila vs.
Anopheles
Mfld (

5. Figure 2. Bantam Encodes a miRNA
3rd instar larval RNA (Northern)
21nt
584nt
100nt
in 3’UTR
of UAS-EGFP
S1 nuclease –protection
mapping

6. Figure 3. Bantam miRNA expression
Tub—GFP—ban,ban(target)
Tub—GFP—UTR
Upregulation of bantam sensor in
clones of mutant cells
Downregulation of bantam sensor
in clones overexpressing bantam
Mutant clones smaller in size
than wild-type twins;
bantam acts cell autonomously

7. Fig.4 bantam Expression and Cell Proliferation
bantam sensor = green
BrdU incorporation - purple
Proliferating cells are correlated with increased bantam miRNA activity (i.e. decreased sensor)
Larval brain
Wing disc (ZNC = Zone of Non-proliferating Cells)
ptc-Gal4 x UAS-C + GFP = green
i.e. bantam expression correlates
with cell proliferation

8. Fig.5 bantam inhibits proliferation-induced apoptosis
studies on Myc and E2F oncogenes have shown that strong proliferative stimuli induce apoptosis;
Cell proliferation results only when apoptosis is simultaneously prevented.
Can bantam suppress proliferation-induced apoptosis caused by overexpression of E2F
and its cofactor DP?
Activated caspase 3
DAPI-labeled nuclei
+ bantam
Expansion of proliferation; reduction in PCD

9. Fig.6 bantam regulates the hid 3’UTR
5 bantam target sites
in melanogaster and
pseudoobscura
Tubulin-EGFP-hid3’UTR = green
Wg protein = red
bantam expression from
EP(3)3622

10. Fig. 7 bantam Regulates Hid Expression
Hid antibody
AS-RNA probe
for hid mRNA
Activated caspase 3
antibody
ptc>hid
ptc>hid
+ bantam
…at the level of protein
…and blocks the apoptosis-inducing effects of hid

11. Fig. 8 bantam levels regulate hid activity in eye development